Mammographic breast density is a strong predictor of breast cancer risk. Women with radiodense areas[unreadable] covering greater than 50% of the tissue area have a 3 to 5 fold increase in risk for breast cancer compared[unreadable] with women with little or no radiodense area. It is estimated that almost 1/3 of breast cancer incidence is[unreadable] due to biological variables (both genetic and environmental) that modulate breast density. Given this[unreadable] strong correlation between breast density and risk for breast cancer, it is surprising that so little is known[unreadable] about the character or origin of dense breast tissue.[unreadable] We hypothesize that the molecular interactions between stroma and epithelium represent some of[unreadable] the very first changes that occur within breast tissue allowing malignant transformation and may therefore[unreadable] serve as a predictor of breast cancer in its earliest stages. We propose that features of increased stromal[unreadable] remodeling noted in the extracellular matrix (ECM) and molecular markers in mammographically dense[unreadable] breasts are indicative of an 'activated' stroma. Activated stroma (AS) is similar to stroma formed during[unreadable] normal, non-pathological processes such as morphogenesis and wound healing, and can also be found in[unreadable] pathological states such as desmoplasia. While normal stromal-epithelial interactions actively suppress[unreadable] preneoplastic phenotypes, activated stroma can become an active participant in cancer progression. We[unreadable] hypothesize that the mechanistic links between high breast density and increased breast cancer[unreadable] risk lie in the signal transduction pathways that lead to increased breast density and[unreadable] concomitantly promote malignant progression in initiated cells in the adjacent epithelium. This[unreadable] proposal will examine several aspects of the phenotypic, molecular and functional differences of[unreadable] mammary fibroblasts and epithelial cells isolated from individuals with high or low mammographic density[unreadable] that have or have not developed breast cancer. Histologic and molecular evaluation of these tissues will[unreadable] provide novel markers that define increased breast density and increased cancer risk. In Project 2, we will[unreadable] 1) Determine the cellular and histological composition of human breast tissues with high and low[unreadable] mammographic density. A three-dimensional reconstruction (3D) of the gland, linked to the BioSig[unreadable] database, will integrate this morphologic data with molecular data. (2) Using cDNA microarrays, we will[unreadable] compare expression profiles from tissues with high and low mammographic densities (a) to each other[unreadable] and (b) to matched tissues from individuals that have developed cancer at a distant site. These markers[unreadable] can be used in Project 3 to evaluate paraffin-preserved benign breast biopsy tissue by immunostains for[unreadable] association of risk for breast cancer. In Specific Aim 3 In vivo and in vitro recombinant experiments will[unreadable] provide insights into the cell combinations that generate increased breast density and molecular markers[unreadable] associated with increased breast cancer risk. (3) Finally we will determine the functional phenotype of[unreadable] fibroblast cells obtained from human breast tissues with high and low mammographic density. We have[unreadable] found that fibroblasts from tissue with high mammographic density retain differential expression of[unreadable] biologically relevant pathways such as the IGF-axis. These fibroblasts have been demonstrated to[unreadable] facilitate tumor progression when placed in a murine recombinant model. The proposed studies will[unreadable] determine the molecular basis for fibroblast enhancement of tumorigenic phenotypes.